The invention relates to DNA vaccines.
Antigen-specific immunotherapy has recently emerged as an approach for controlling cancer because it is capable of developing specific immunity against neoplastic cells while not attacking normal cells. DNA vaccination differs from traditional vaccination in that DNA encoding an antigen (not the antigen itself) is injected into the subject. The production of the antigen, i.e., expression of the antigen encoded by DNA in the vaccine, takes place in the body of the vaccinated individual. However, conventional DNA vaccines have limitations. For example, a major drawback of most DNA vaccines is their potency.
The invention is based on the discovery that the immunogenicity of a target antigen encoded by a DNA vaccine is enhanced by the presence in the DNA vaccine construct of DNA encoding a carboxyterminal portion of Mycobacterium tuberculosis heat shock protein 70 (HSP70). Accordingly, the invention provides compositions and methods of vaccination that significantly enhance the potency, and thus clinical efficacy, of DNA vaccines.
An immunogenic composition contains a first DNA encoding a carboxyterminal fragment of a heat shock protein, e.g., HSP70, operably linked to a second DNA encoding a MHC class I restricted antigen. A carboxyterminal fragment of a protein is a polypeptide which is at least 10 amino acids in length and is derived from a half of a naturally-occurring protein that contains a COOH end. For example, a carboxyterminal fragment of HSP70 is a peptide the amino acid sequence of which is derived from a portion of HSP70 spanning residues 312-625 of SEQ ID NO:9 and is encoded by DNA spanning the coding region of those residues. Preferably, the carboxyterminal fragment contains the amino acid sequence of residues 517-625 of SEQ ID NO:9. For example, the immunogenic composition contains a first DNA encoding a polypeptide containing the amino acid sequence of residues 517 to 625 of SEQ ID NO:9 operably linked to a second DNA encoding a MHC class I restricted antigen. The invention therefore includes an E7-HSP70-CD fusion polypeptide as well as DNA encoding the fusion polypeptide. Optionally, the composition includes DNA encoding a polypeptide containing residues derived from the aminoterminal fragment of HSP70, e.g., residues 161-370 of SEQ ID NO:9. The order in which the DNA components, e.g, first and second (and optionally, third), DNAs are operably linked can be altered without affecting immunogenicity. For example, the HSP-encoding DNA sequences are located 5xe2x80x2 or 3xe2x80x2 to the target antigen-encoding sequences. Preferably, the DNA is operably linked so that the DNA construct encodes a recombinant polypeptide in which the MHC class I restricted antigen is located aminoterminal to the HSP-derived residues.
As is discussed below, the MHC class I restricted antigen is derived from a pathogen such as a virus or from a cancer tissue. The DNA vaccine of the invention contains a plasmid vector, which contains a first DNA encoding a carboxyterminal fragment of a heat shock protein operably linked to a second DNA encoding a MHC class I restricted antigen. Therapeutic methods include methods of inducing a cytotoxic T cell response to an antigen in a mammal by administering to the mammal the immunogenic compositions described herein.
Alternatively, an immunogenic composition of the invention contains the following components: (a) a first DNA containing a sequence encoding a polypeptide which binds to a professional antigen presenting cell, (b) a second DNA containing a sequence encoding a cytoplasmic translocator polypeptide, and (c) a third DNA containing a sequence encoding a major histocompatibility complex (MHC) class I restricted antigen. The first, second, and third DNAs are operably linked. By xe2x80x9coperably linkedxe2x80x9d is meant that DNA encoding a polypeptide is joined in frame to another DNA encoding a another polypeptide. Translation of operably linked DNAs yields a chimeric polypeptide or fusion gene product. The order in which the first, second, and third DNAs are operably linked is irrelevant. The construct may also contain regulatory sequences. A polypeptide coding sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
The first DNA of the immunogenic composition encodes a fragment of a heat shock protein (HSP), e.g., Mycobacterium tuberculosis heat shock protein 70 (HSP70). A xe2x80x9cfragmentxe2x80x9d of a given protein is a polypeptide which is shorter in length than the reference polypeptide. For example, the polypeptide is at least 9 or 10 amino acids in length but less than the total number of residues of the mature reference protein or polypeptide. For example, a fragment of HSP70 is less than 70 kDa in molecular mass. A fragment of HSP70 has an amino acid sequence that is at least 50% identical to the amino acid sequence of a naturally-occurring HSP70. The length of an HSP70 fragment is less than 625 amino acids. Preferably, the fragment has a biological activity of the reference protein. For example, the fragment binds to a professional antigen presenting cell (APC) such as a dendritic cell (DC), or the fragment mediates translocation into the cytoplasm of the chimeric polypeptide encoded by the DNA of the immunogenic composition. In some embodiments, the first DNA or the second DNA (or both) encode a fragment of a heat shock protein.
The third DNA of the immunogenic composition encodes an antigenic epitope such as a MHC class I-restricted antigen. Preferably, the antigen is derived from a virus such as a human papovavirus, e.g., a cervical cancer-associated human papillomavirus (HPV). The viral antigen is all or a part of E6 or E7 antigen derived from HPV-16. Other viral antigens include the E2 antigen of bovine viral diarrhea virus; ppUL83 or pp89 of cytomegalovirus; prM/E of encephalitis virus SLE; HBV surface antigen or HBV core antigen of hepatitis B virus; HIV-1 antigens such as gp160; ICP27, gD2, glycoprotein B, or glycoprotein B of herpes simplex virus. Alternatively, the antigen is a cancer antigen such as a mutant p53; MAGE-1 or MAGE-3 associated with melanoma cells, e.g. a cancer-associated antigen expressed on the surface of a tumor cell. Other antigens include malaria peptide (NANP)40, HIV-1 p24, or influenza nucleoprotein.
In one example, the first DNA encodes granulocyte-macrophage colony stimulating factor (GM-CSF) or a fragment thereof. Preferably, a functional GM-CSF fragment contains a disulfide bridge, e.g., a disulfide bridge which spans Cys 51 and Cys93 of SEQ ID NO:1. Other biologically active fragments of GM-CSF are polypeptides which include residues 18-22, 34-41, 38-48, 52-61, 94-115, 95-111 of SEQ ID NO:1. Alternatively, the first DNA (encoding an APC-binding polypeptide) encodes Flt3 ligand (FL), CTLA-4, 4-1BB, CD40 ligand, or TNF receptor (or an APC-binding fragment thereof).
The second DNA may encode a translocation domain of a Pseudomonas exotoxin A (ETA), e.g,. domain II (dII) of ETA (spanning residues 253-364 of SEQ ID NO:3). A translocation domain is a polypeptide that induces translocation of protein or polypeptide to which it is linked into the cytosol of a cell. For example, the second DNA encodes a polypeptide derived from a Diphtheria, clostridial (Botulinum, Tetanus), Anthrax, Yersinia, Cholera, or Bordetella pertussis toxin. The presence of DNA encoding a translocation domain in the immunogenic composition enhances MHC class I presentation of the antigen encoded by the composition through translocation of antigen from the endosomal/lysosomal compartment to the cytosol. Preferably, the toxic domain of the gene encoding the toxin is mutated or deleted.
The immunogenic composition need not contain a first, second, and third DNA, as described above. In one alternative embodiment, the third DNA (encoding a target antigen to which immunity is desired) is operably linked to a DNA encoding an APC binding polypeptide (in the absence of DNA encoding a translocator polypeptide); in antoh or a second DNA encoding a translocator polypeptide. For example, the DNA encodes a fusion polypeptide containing E7-ETA, E7-HSP70, E7-GM-CSF, or E7-FL. The immunogenic composition contains DNA encoding residues 1-189 of SEQ ID NO:25 operably linked to DNA encoding an antigen to which immunity is sought.
The immunogenic composition includes DNA encoding an E7-HSP70 fusion polypeptide, DNA encoding a GM-ETA(dII)-E7 fusion polypeptide, or DNA encoding ETA(dII)-E7 (i.e., without the GM component).
Also within the invention is a DNA vaccine. The DNA vaccine composition contains a plasmid vector which includes (a) a first DNA encoding a polypeptide which binds to a professional antigen presenting cell, (b) a second DNA encoding a cytoplasmic translocator polypeptide, and (c) a third DNA encoding a MHC class I restricted antigen. The DNAs are cloned into the plasmid vector in such a way that the first, second, and third DNAs are operably linked. When transcribed and translated in the cell in which the plasmid vector has been taken up, the vector directs production of a chimeric polypeptide which includes an APC-binding portion, a cytoplasmic translocator portion, and an epitope of a class I-restricted antigen.
The DNAs or polynucleotides of the invention are isolated. By xe2x80x9cisolatedxe2x80x9d is meant a nucleic acid molecule that is free of the genes which, in the naturally-occurring genome of the organism, flank the gene sequence of interest. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a procaryote or eucaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. The term excludes large segments of genomic DNA, e.g., such as those present in cosmid clones, which contain a given DNA sequence flanked by one or more other genes which naturally flank it in a naturally-occurring genome.
Nucleic acid molecules (or polynucleotides) include both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Where single-stranded, the nucleic acid molecule may be a sense strand or an antisense strand. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a procaryote or eucaryote at a site other than its natural site; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
The DNAs of the immunogenic composition or DNA vaccine contain a strand which has the nucleotide sequence of a given reference sequence or which hybridizes at high stringency to a strand of DNA having the reference sequence, or the complement thereof. For example, the DNA has at least 50% sequence identity to a reference sequence, and encodes a polypeptide having a specified biological activity. For example, the biological activity of the polypeptide encoded by the first DNA of the immunogenic composition is binding to an APC, the biological activity of the peptide encoded by the second DNA is cytoplasmic translocation, and the biological activity of the peptide encoded by the third DNA is binding to a MHC class I molecule. Preferably, the DNA has at least 75% identity, more preferably 85% identity, more preferably 90% identity, more preferably 95% identity, more preferably 99% identity, and most preferably 100% identity to the reference sequence.
Nucleotide and amino acid comparisons are carried out using the Lasergene software package (DNASTAR, Inc., Madison, Wis.). The MegAlign module used was the Clustal V method (Higgins et al., 1989, CABIOS 5(2):151-153). The parameter used were gap penalty 10, gap length penalty 10.
Alternatively, the DNAs of the immunogenic composition hybridize at high stringency to a strand of DNA having the reference sequence, or the complement thereof and encode a polypeptide with a specific biological activity. Hybridization is carried out using standard techniques, such as those described in Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, 1989). xe2x80x9cHigh stringencyxe2x80x9d refers to nucleic acid hybridization and wash conditions characterized by high temperature and low salt concentration, i.e., wash conditions of 65xc2x0 C. at a salt concentration of approximately 0.1xc3x97SSC. xe2x80x9cLowxe2x80x9d to xe2x80x9cmoderatexe2x80x9d stringency refers to DNA hybridization and wash conditions characterized by low temperature and high salt concentration, i.e., wash conditions of less than 60xc2x0 C. at a salt concentration of at least 1.0xc3x97SSC. For example, high stringency conditions may include hybridization at about 42xc2x0 C., and about 50% formamide; a first wash at about 65xc2x0 C., about 2xc3x97SSC, and 1% SDS; followed by a second wash at about 65xc2x0 C. and about 0.1%xc3x97SSC. Lower stringency conditions suitable for detecting DNA sequences having about 50% sequence identity to a reference gene or sequence are detected by hybridization at about 42xc2x0 C. in the absence of formamide;. a first wash at about 42xc2x0 C., about 6xc3x97SSC, and about 1% SDS; and a second wash at about 500C, about 6xc3x97SSC, and about 1% SDS.
The invention includes a method of inducing a cytotoxic T cell response to an antigen in a mammal by administering to the mammal the immunogenic composition described above. In preferred embodiments, the composition is administered as naked DNA. The DNA is also administered in the presence of agents which enhance uptake of the DNA by target cells, such as phospholipid formulation, e.g, a liposome. The method is useful to vaccinate a mammal against infection by a pathogen; in this case, the third DNA encodes an antigen derived from said pathogen such as a virus or bacteria. The method is also useful to prevent the development of cancer or treat an existing cancer in a mammal. Mammals at risk of developing a certain type of cancer are identified using known methods, e.g. genetic screening. Individuals at risk of developing a cancer or suffering from cancer are treated by administering the composition in which the third DNA encodes a cancer-associated antigen such as HPV E7 which is associated with cervical cancer.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.